Sensitivity and specificity of monoclonal and polyclonal immunohistochemical staining for West Nile virus in various organs from American crows (Corvus brachyrhynchos).

BACKGROUND: Based on results of earlier studies, brain, heart and kidney are most commonly used for West Nile virus (WNV) detection in avian species. Both monoclonal and polyclonal antibodies have been used for the immunohistochemical diagnosis of WNV in these species. Thus far, no studies have been performed to compare the sensitivity and specificity of monoclonal and polyclonal antibodies in detecting WNV in American crows (Corvus brachyrhynchos). Our objectives were to determine 1) the comparative sensitivities of monoclonal and polyclonal antibodies for immunohistochemical (IHC) diagnosis of WNV infection in free-ranging American crows, 2) which organ(s) is/are most suitable for IHC-based diagnosis of WNV, and 3) how real-time RT-PCR on RNA extracted from formalin-fixed paraffin-embedded tissues compared to IHC for the diagnosis of WNV infection. METHODS: Various combinations, depending on tissue availability, of sections of heart, kidney, brain, liver, lung, spleen, and small intestine from 85 free-ranging American crows were stained using a rabbit-polyclonal anti-WNV antibody as well as a monoclonal antibody directed against an epitope on Domain III of the E protein of WNV. The staining intensity and the extent of staining were determined for each organ using both antibodies. Real-time RT-PCR on formalin-fixed paraffin-embedded tissues from all 85 crows was performed. RESULTS: Forty-three crows were IHC-positive in at least one of the examined organs with the polyclonal antibody, and of these, only 31 were positive when IHC was performed with the monoclonal antibody. Real-time RT-PCR amplified WNV-specific sequences from tissue extracts of the same 43 crows that were IHC-positive using the polyclonal antibody. All other 42 crows tested negative for WNV with real-time PCR and IHC staining. Both antibodies had a test specificity of 100% when compared to PCR results. The test sensitivity of monoclonal antibody-based IHC staining was only 72%, compared to 100% when using the polyclonal antibody. CONCLUSION: The most sensitive, readily identified, positively staining organs for IHC are the kidney, liver, lung, spleen, and small intestine. Real-time RT-PCR and IHC staining using a polyclonal antibody on sections of these tissues are highly sensitive diagnostic tests for the detection of WNV in formalin-fixed tissues of American crows.

Molecular epidemiology and cluster analysis of human listeriosis cases in three U.S. states.

To better understand the transmission and epidemiology of human listeriosis, 647 Listeria monocytogenes isolates obtained from human listeriosis cases in four U.S. locations (Michigan, Ohio, New York State, and New York City) over 61 months (1998 to 2003) were characterized by automated EcoRI ribotyping. A total of 65 ribotypes were differentiated among the characterized isolates; 393, 227, and 24 isolates were classified into lineages I, II, and III, respectively, and 3 isolates were not classified to lineage. The three most common ribotypes (responsible for 39% of all cases) represented L. monocytogenes epidemic clones, each of which had previously been linked to at least two human listeriosis outbreaks. Categorical analyses revealed that ribotypes and lineages were nonrandomly distributed among the four locations. Temporal cluster analysis of cases identified 13 statistically significant temporal subtype clusters, which represented 26% of all cases. Three of these clusters matched previously described human listeriosis outbreaks. Isolates involved in clusters belonged to nine ribotypes. Four, eight, and one cluster were caused by lineages I, II, and III, respectively. The two largest clusters were both caused by the epidemic clone representing ribotype DUP-1044A. Categorical analyses revealed no significant associations between lineage or ribotype and clinical manifestation (central nervous system infection, septicemia, fetal infection, or other infection) or disease outcome (fatal or not fatal). Although human listeriosis cases are caused by isolates belonging to a diversity of EcoRI ribotypes, specific lineage I epidemic clones cause a large number of human listeriosis cases. Many human listeriosis cases can be grouped into statistically significant temporal clusters, including widely distributed and region-specific clusters associated with isolates of various ribotypes. L. monocytogenes lineages and EcoRI ribotypes do not appear to differ in their likelihood of causing different clinical manifestations or mortality.

An outbreak of bronchoscopy-related Mycobacterium tuberculosis infections due to lack of bronchoscope leak testing.

BACKGROUND: Bronchoscopy-related transmission of Mycobacterium tuberculosis is rarely reported. In August 1999, five M tuberculosis-positive bronchial washing culture findings were noted in patients who underwent bronchoscopy in July in a hospital that reported only eight M tuberculosis-positive culture findings from 1995 to 1998, prompting further investigation. METHODS: A case was defined as a M tuberculosis-positive culture finding from specimens obtained from patients who underwent bronchoscopy during January to August of 1999. Bronchoscopy and laboratory records, procedures, and practices were reviewed. M tuberculosis isolates were compared using restriction fragment length polymorphism (RFLP) analysis. RESULTS: During July 1999, 19 bronchoscopic procedures were performed in 19 patients. Bronchial washing specimens for mycobacterial culture were obtained from 18 patients. Ten cases were identified. Two case patients, including the index patient, had signs and symptoms of active tuberculosis prior to bronchoscopy. M tuberculosis infections developed in two more case patients despite starting a standard four-drug antituberculous regimen within 3 weeks after bronchoscopy. Six case patients had positive culture findings but no evidence of infection. All M tuberculosis isolates were antituberculosis-drug susceptible, and all but one were indistinguishable by RFLP analysis. Three bronchoscopes were used during the outbreak period; one bronchoscope was used in 9 of the 10 case patients (relative risk, 8.1; 95% confidence interval, 1.3 to 52). A hole was discovered in the sheath of this bronchoscope. Leak testing, a critical step in bronchoscope reprocessing, was not routinely performed at this institution. CONCLUSIONS: M tuberculosis contamination of the bronchoscope occurred during the index patient's procedure. The hole in the sheath provided access to a space that was difficult to mechanically clean and chemically disinfect. The reprocessing recommendations of bronchoscope manufacturers, including leak testing after each use, should be closely followed.

Persistence of West Nile Virus (WNV) IgM antibodies in cerebrospinal fluid from patients with CNS disease.

The Michigan Department of Community Health (MDCH) reported 644 laboratory positive human cases of West Nile Virus (WNV) in the 2002 outbreak in the US, of which 559 cases presented with either meningitis or encephalitis. The first line test utilized for diagnosis of WNV infection was the immunoglobulin M (IgM)-capture enzyme-linked immunosorbent assay (MAC-ELISA). We continued testing for WNV even during winter months of the year 2002-2003 due to the awareness of other modes of WNV transmission (blood transfusion, organ transplantation, transplacental, breast milk, and occupational) as well as concern for people traveling to endemic areas. As a result of year-round testing for WNV infections during 2002-2003, we detected WNV IgM-specific antibodies in cerebrospinal fluid (CSF) specimens from three patients persisting for 110, 141, and 199 days post acute phase infection in patients with central nervous system (CNS) disease. This is a new observation and there is no published data on the persistence of WNV IgM antibodies in CSF specimens beyond 47 days. Thus, it is important to note that the presence of WNV IgM class antibodies may not always reflect acute phase infection with this virus.

Epidemiologic investigation of Mycobacterium bovis in a population of cats.

OBJECTIVE: To determine whether cats exposed at a residence were infected with Mycobacterium bovis, whether the tuberculin skin test can identify cats infected with M bovis, and whether an ELISA could identify tuberculosis-infected cats. ANIMALS: 20 domestic cats exposed to a cat with laboratory-confirmed disseminated M bovis infection. PROCEDURE: Cats were administered a tuberculin skin test and monitored for 72 hours. Blood and fecal samples were collected. Cats were then euthanatized, and postmortem examinations were performed. Tissues were examined grossly and histologically for signs of mycobacteriosis. Pooled tissue samples and fecal samples were submitted for mycobacterial culture. Blood samples were examined for evidence of tuberculosis by use of a comparative ELISA. RESULTS: 4 cats had positive responses for the ELISA, and 2 cats had suspicious responses. All tuberculin skin tests yielded negative results. No gross or histologic lesions of tuberculosis were detected in any tissues, and mycobacteria were not isolated from tissues or feces obtained from the 20 cats. CONCLUSIONS AND CLINICAL RELEVANCE: All cats that had positive or suspicious responses for the ELISA were offspring of the cat with tuberculosis. Evidence of tuberculosis was not seen in other cats at the residence, the owner, or the attending veterinarian. The most likely source of tuberculosis for the infected cat was through the consumption of M bovis-infected wildlife carcasses or offal. Because M bovis is endemic in wildlife in northeastern Michigan, there is a risk of exposure to tuberculosis in companion animals, their owners, and attending veterinarians.

Variable-number tandem repeat typing of Mycobacterium tuberculosis isolates with low copy numbers of IS6110 by using mycobacterial interspersed repetitive units.

A study set of 180 Mycobacterium tuberculosis and Mycobacterium bovis isolates having low copy numbers of IS6110 were genotyped using the recently introduced method based on the variable-number tandem repeats of mycobacterial interspersed repetitive units (MIRU-VNTR). The results were compared with results of the more commonly used methods, IS6110 restriction fragment length polymorphism (RFLP) and spoligotyping. The isolates were collected in Michigan from 1996 to 1999 as part of a project to genotype all isolates from new cases of tuberculosis in the state. Twelve MIRU loci were amplified, and the amplicons were analyzed by agarose gel electrophoresis to determine the copy number at each MIRU locus. MIRU-VNTR produced more distinct patterns (80 patterns) than did IS6110 RFLP (58 patterns), as would be expected in this study set. Spoligotyping identified 59 patterns. No single method defined all unique isolates, and the combination of all three typing methods generated 112 distinct patterns identifying 90 unique isolates and 90 isolates in 22 clusters. The results confirm the potential utility of MIRU-VNTR typing and show that typing with multiple methods is required to attain maximum specificity.

Purification of Sarcocystis neurona sporocysts from opossum (Didelphis virginiana) using potassium bromide discontinuous density gradient centrifugation.

This report describes a new, inexpensive procedure for the rapid and efficient purification of Sarcocystis neurona sporocysts from opossum small intestine. S. neurona sporocysts were purified using a discontinuous potassium bromide density gradient. The procedure provides a source of sporocyst wall and sporozoites required for reliable biochemical characterization and for immunological studies directed at characterizing antigens responsible for immunological responses by the host. The examined isolates were identified as S. neurona using random amplified polymorphic DNA primers and restriction endonuclease digestion assays. This method allows the collection of large numbers of highly purified S. neurona sporocysts without loss of sporocyst viability as indicated by propidium iodide permeability and cell culture infectivity assays. In addition, this technique might also be used for sporocyst purification of other Sarcocystis spp.